GFP's importance was recognized with the 2008 chemistry Nobel Prize. In 2007 more than 12,000 fluorescent protein (FP) papers were published, the majority used GFP as a molecular probe. A surprisingly small number examined the structural and photophysical properties of the FPs. The research proposed here is sub-divided into smaller units that can be completed by individual students or teams of students in two to three years of undergraduate research. 1. Photophysics: Dihedral Freedom of the Chromophore Using molecular dynamics simulations with freely rotating chromophoric dihedrals, we will examine the conformational freedom of the chromophore in red fluorescent and highlighter proteins. Is dihedral freedom related to the fluorescence quantum yield? 2. Chromophore Cavity and Photophysical Properties of FPs Relibase+ will be used to find proteins that have cavities that can bind GFP-chromophore model compounds and to examine the evolution of the chromophore cavity in GFP-like proteins. 3. Proton Transfer Chains in FPs The proton-wires are well-defined in the FP crystal structures. Therefore the proton-wires in FPs are not only important in understanding the photophysical behavior of the FPs, but also as a model for proton transfer in other proteins with less well-defined proton-wires. Chemometric and molecular modeling techniques will be used to determine the rigidity of the proton wires, and to find alternative paths for proton transfer. 4. Searching For Bioluminescent Proteins in the Global Ocean Sampling Expedition Database Bioinformatic and homology modeling of FPs and luciferases in GOS. 5. Computational Evolution of Fluorescent Nidogen The G2 nidogen domain is unexpectedly similar to the 2-barrel of FPs. We will use our knowledge of chromophore formation in GFP to design a nidogen mutant that will autocatalytically cyclize to form a chromophore. 6. Integrity of the 2-Barrel Activated molecular dynamics will be used to find the paths available to water molecules in and out of GFP, its mutants and homologs. 7. GFP Educational Outreach GFP will be used to excite young students and non- scientists about science. Through the popular press, web, school visits and teacher workshops we especially hope to reach out to underrepresented students. PUBLIC HEALTH RELEVANCE: Computational methods will be used to improve the understanding and design of GFP-like proteins. In the last fifteen years green fluorescent protein (GFP) has changed from a nearly unknown protein to a commonly used molecular imaging tool in biology, chemistry, genetics and medicine. The palette of colors available to researchers wanting to use GFP has been expanded by mutating GFP and by finding new GFP-like proteins in marine organisms, yet there is still much to be learned in the FP field.